The object of this research is to assess the importance of damage to transcribing and nontranscribing DNA sequences for the survival of the mammalian cell. Two different approaches are proposed. In the first we determine whether cells surviving DNA damage have more damage in non-transcribing sequences than transcribing sequences. Cells will be UV-irradiated and the surviving cells harvested at various times. The chromatin is then fractionated and the level of damage assayed in each fraction. An apparently differential loss of damage with time from transcribing DNA could be due to one of two factors - selective survival or preferential repair of this DNA. This will be tested by quantitating the level of repair replication in each fraction. The second approach exploits the fact that there is preferential replication of transcribing sequences in early S phase. The effect on survival of BUdR pulses given at various times throughout S phase will be determined. In addition, a second identical batch of cells will be pulse-labeled with 3H-TdR and the chromatin fractionated. In this way we will have a direct estimate as to the time of replication in S phase of transcribing and non-transcribing sequences, and in addition, we will determine whether BUdR incorporation into transcribing DNA is more damaging to the cell. We have evidence that BUdR incorporation effects the order of replication of DNA sequences and we propose to determine whether the detrimental effect of BUdR incorporation into DNA is mediated through this effect or results directly from DNA damage. The latter can be enhanced by irradiating cells containing BUdR with 313 nm light. For similar levels of survival (with and without irradiation) the effects on the pattern of DNA replication can be compared.